Role of gene interactions in the pathophysiology of skeletal dysplasias: A case report in Colombia

Background Genome association studies have shown that gene-gene interactions or epistasis play a crucial role in identifying the etiology, prognosis, and treatment response of many complex diseases beyond their main effects. Skeletal dysplasias are a heterogeneous group of congenital bone and cartilage disorders with a genetic and gen-gen interaction etiology. The current classification of skeletal dysplasias distinguishes 461 diseases in 42 groups, and the incidence of all skeletal dysplasias is more than 1 in every 5000 newborns. The objective is to present the case of a patient with four variants that generates gen-gen interactions in the skeletal dysplasia. Case presentation A 1-year-old male patient was diagnosed with skeletal dysplasia based on prenatal ultrasound showing micromelia and pyelocalyceal dilation. Postnatal physical examination revealed body disproportion and involvement of other organs and systems. Materials and Methods A sequencing study and deletions/duplications analysis were performed for 358 candidate genes associated with skeletal dysplasia. The GeneMANIA interface was used to evaluate the expression network of genes associated with each other for the gen-gen interaction. Results Four pathogenic variants were obtained two heterozygous variants with pathogenic significance in SLC26A, one heterozygous pathogenic variant in CLCN7 and another heterozygous pathogenic variant in CEP120. The GeneMANIA interface reveals 77.64% physical interactions, 8.01% co-expression, 5.37% prediction, 3.63% co-localization, 2.87% genetic interactions, 1.88% route of action, and 0.60% shared protein domains. Discussion and Conclusions These results suggest that the interaction between these genes affects the activity of the inorganic anion exchanger, leading to disorganization of collagen fibers, early mineralization, and decreased assembly of fibronectin in the bone extracellular matrix. Identifying gene-gene interactions is a fundamental step in understanding proper cell function and thus understanding the pathophysiology of many complex human diseases, improving diagnosis, and the possibilities of new personalized therapies.

][3][4][5] Gene variations resulting from epistasis can affect more than one system.In the case of the musculoskeletal system, patients are characterized by short stature, impaired ossification, gait, and orthopedic problems, known as musculoskeletal dysplasia (SD, Skeletal dysplasia).[8][9][10][11] In the latest epidemiological studies worldwide, it has been reported that the incidence of SD is from 2.3 to 7.6 per 10,000 live births, of these, it is known that osteogenesis imperfecta (OI), the most common pathological entity of SD, is 1 in 15,000 live births worldwide.However, the true worldwide incidence rate may be higher since SD encompasses both viable and life-limiting fatal disorders.][13][14][15][16][17] The objective is to present the case of a patient with four variants that generates gen-gen interactions in the skeletal dysplasia.

Consent
After obtaining consent from the mother, we got approval from the medical ethical committee, in order to access the clinical history of the patient to make the present case report.

Clinical evaluation
A 1-year-old male patient, son of non-consanguineous parents and with no family history of skeletal dysplasias, who in the prenatal stage presented an anatomical size ultrasound at week 20 of gestation showing micromelia and pyelocalyceal dilation.At birth, the patient was found with body disproportion with shortening of the upper and lower limbs more proximal than distal, equinovarus foot, ligamentous laxity and dysmorphic facies, given by epicanthal folds, retrognathia, high palate, low-set lobed ears.Ophthalmological and auditory evaluation without alterations, at the cardiovascular level with transthoracic echocardiogram showing restrictive patent foramen ovale with aneurysmal septum primum to the right, normal LVEF systolic function 67 %.Long bones x-ray with preserved bone density, without traces of fractures or lytic or blastic lesions.
An X-ray of the upper limbs was performed, showing thickened and shortened long arm bones and forearms, without alteration of the hand bones.Ultrasound of the abdomen showed pyelocalyceal dilatation of the left kidney, bilateral hip ultrasound with grade II bilateral hip dysplasia, and X-ray of the lower limbs showed clubfoot, which was corrected with surgical management and a Dennis Brown splint.

Methods
To investigate the genetic causes of the clinical manifestations, we performed sequencing and deletion/duplication analysis on 358 candidate genes using genomic DNA obtained from both prenatal and postnatal samples, We used a protocol based on hybridization to enrich the genomic DNA obtained from submitted samples for target regions.] We sequenced all target regions at a depth of at least 50x, or performed additional analyses to ensure sufficient coverage.We aligned the resulting reads to the GRCh37 reference sequence and identified sequence changes in the context of a single clinically relevant transcript.
We focused our enrichment and analysis on the coding sequence of the indicated transcripts, as well as 10 bp of flanking intronic sequence (20 bp for BRCA1/2) and other specific disease-causing genomic regions identified at the time of assay design.We did not evaluate promoters, untranslated regions, or other non-coding regions, and for some genes, only specific target loci were analyzed.
We identified exonic deletions and duplications using an internal algorithm that compares the read depth of each target in the proband sequence to the mean read depth obtained from a set of clinical samples, in order to determine the number of copies in each target.
Markers on the X and Y chromosomes were analyzed for quality control purposes and to be able to detect deviations from the expected sex chromosome complement.All clinically significant observations were confirmed using additional testing methods, except for variants that were already confirmed in a first-degree relative or had been individually validated.

Result
Four pathogenic variants were obtained two heterozygous variants with pathogenic significance in SLC26A, one heterozygous pathogenic variant in CEP120 and one heterozygous pathogenic variant in CEP120, Table 1.
GeneMANIA interface led to obtain the network of genes associated with each other in the patient, Illustration.1, in which 77.64 % physical interactions, 8.01 % co-expression, 5.37 % prediction, 3.63 % colocalization, 2.87 % genetic interactions, 1.88 % pathway of action and 0.60 % shared protein domains.

Discussion
SDs, also known as osteochondrodysplasias, are a group of heterogeneous disorders associated with bone and cartilage development; sometimes accompanied by pulmonary, genitourinary, visual, auditory, and neurodevelopmental manifestations.In 2019, the International Society for Skeletal Dysplasia published an updated version of the SD nosology, which includes 461 skeletal genetic disorders and identifies 437 different causative genes grouped into 42 categories based on their clinical, radiographic, and/or molecular characteristics.This comprehensive classification system highlights the challenges associated with diagnosing SDs due to their heterogeneous nature. 7,9,11D are considered multiple congenital anomalies syndromes and have been linked to genes variants encoding components of RNA degradation, although we are currently unaware of a human disease caused by variants in a ribonuclease that specifically targets messenger RNA polyadenylation (poly(A)), which leads to the phenotype varying in severity.] A characteristic feature of the SD phenotype is body disproportion due to the shortening of the limbs, trunk, or both.] In recent decades, efforts have been made to elucidate the genetic pathophysiology of SD, which has allowed the emergence of new therapeutic approaches such as cell therapy, gene therapy, or drug therapy for various conditions.Several therapeutic strategies are currently being investigated in osteogenesis imperfecta.There are ongoing clinical trials based on pharmacological approaches, targeting signaling pathways in specific types of skeletal dysplasias, such as achondroplasia and Fibrodysplasia ossificans progressiva (FOP), or addressing endoplasmic reticulum stress in conditions such as Schmid-type metaphyseal dysplasia or pseudoachondroplasia.In addition, the treatment of hypophosphatasia, or Morquio A disease, illustrates the efficacy of enzyme drug replacement.These diseases require highly specialized and multidisciplinary approaches involving various specialists, such as geneticists, orthopedic surgeons, and endocrinologists.The emergence of treatments for skeletal dysplasia provides new insights into the prognosis of these severe conditions and may change prenatal advice for these diseases in the coming years. 11he patient described carried a compound heterozygous variant in the SLC26A2 gene, also known as the "solute carrier family 26 members 2" gene, located in sub-band 2 of band 3 of the long arm of chromosome 5 (5q32).The SLC26A2 gene encodes for a transmembrane glycoprotein that has several functions.It transports inorganic cations/ anions and amino acids/oligopeptides across the cell membrane.It catalyzes the sulfonation of small molecules in the cytosol.It is involved in the activity of the secondary active sulfate transmembrane transporter and the transmembrane transporter of secondary active sulfate.It plays a role in the sulfation of proteoglycans in the extracellular matrix of the musculoskeletal system.][24][25] The presented patient phenotype may clinically match with an intermediate form 26 of diastrophic dysplasia (DTD; OMIM 222600) which previously described in association of compound heterozygous SLC26A2 variants according to what has been previously described by Silveira et al (2023). 27he patient also had a pathogenic heterozygous variant in the CLCN7 gene, which is also known as "voltage-gated chloride channel 7".This gene is located in sub-band 3 of band 13 on the short arm of chromosome 16 (16p13.3)and encodes for the homonymous protein.The product of this gene belongs to the CLC chloride channel family of proteins.Chloride channels play an important role in the plasma membrane and intracellular organelles.Variations in the CLCN7 gene are associated with different forms of osteopetrosis, including autosomal dominant osteopetrosis (ADO), autosomal recessive osteopetrosis (ARO), and the intermediate form.Defects in this gene are the cause of autosomal recessive osteopetrosis type 4 (OPTB4), also called infantile malignant osteopetrosis type 2, as well as the cause of autosomal dominant osteopetrosis type 2 (OPTA2), also known as autosomal dominant Albers-Disease, Schonberg disease, or autosomal dominant Marble disease.A variation in the CLCN7 protein affects the function of osteoclasts, which are cells that break down bone tissue.][30][31] The patient also carries a single heterozygous variant in the CEP120 gene which is associated with recessive diseases and can confer carrier status for conditions known as ciliopathies.CEP120 gene codes for the 120 kDa centrosomal protein, also known as coiled-coil 100 domaincontaining proteins or gene that contains the coiled-coil 100 domain (CCDC100 gene), which is located in sub-band 2, thus band 23 of the long arm of chromosome 5 (5q23.2) and codes for the 120 kDa centrosomal protein, also known as coiled-coil 100 domaincontaining proteins, which is required for centriole elongation from a procentriole which is essential for the formation of centrosomes, cilia, and flagella.Variations on the CEP120 gene lead to Ciliopathies, which are a group of clinical disorders affecting the primary cilium, a 9 + 0 immobile monocilium, and may involve the mobile monocilium or the 9 + 2 motile cilia.][34][35][36][37][38][39][40] Even though kidney disease occurs in up to one third of patients with Joubert syndrome, most commonly in those with variations in CEP120, CEP290, TMEM67, and AHI1.Like other syndromic ciliopathies, the kidney disease can be very variable, going to mild to severe, but since the patient is a carrier, it is unlikely that these gene is the cause.][43] Gen-gen interactions affects the cells by affecting the proteins.In normal gen-gen interactions proteins in the cell membrane interact with each other to affect the activity of transmembrane transporters, which are important groups of proteins that allow the transfer of a specific substance or a group of related substances, from one side of a membrane to the other.Cell membranes and organelle membranes are selectively permeable, meaning they only allow certain molecules to pass through, resulting in limited movement of molecules in and out of cells and organelles.Three main classes of transmembrane transporters have been described in the literature, all of which are integral transmembrane proteins and show a high degree of specificity for the transport of substances.The first class consists of pumps driven by ATP, also known as ATPases, which use the energy of ATP hydrolysis to move ions or small molecules through the membrane to resist gradients or chemical concentration potentials.Secondly, we have channel proteins that form a pore or channel in the membrane, allowing specific molecules or water to pass down their concentration or electrical potential gradients without requiring energy, which makes the process energetically favorable.The third class consists of transporters, which bind to specific molecules and move them across the membrane.5][46] So when a gen variants interacts within each other it generates impaired transmembrane transportation.
GeneMANIA made it possible to show that genes typically do not act alone, but are part of a complex system called the genome, where they interact with other genes modifying the effects of a particular gene or genetic variant.These genetic interactions may be influenced by other genetic elements or external factors.In the present case, the interaction between these genes affects the activity of the inorganic anion exchanger, which normally allows the transfer of a solute or solutes from one side of a membrane to the other, depending on specific factors such as the type of reaction involved, as well as the activity of the transmembrane transporter of the sulfur molecular entity, which allows the transfer of a sulfur-containing molecule, such as sulfates or sulfites, from one side of a membrane to the other.The transmembrane dicarboxylic acid transporter moves dicarboxylic acids into, out of, or within cells or between cells, using a transporter or a pore.Transmembrane carboxylic acid transporter activity enables the transfer of organic acids containing carboxyl groups (COOH) or carboxylate anions (COO-) from one side of a membrane to the other.These compounds play a crucial role in skeletal health and their dysregulation has been linked to skeletal dysplasias and osteopetrosis.[49]

Conclusions
Genetic interactions or epistasis play an important role in determining the risk, progression, and response to treatment of many diseases; therefore, identifying the interactions and how they affect the phenotypes of the different diseases becomes a challenge for current genomics because the variation of traits is determined not only by their genetic components, but also by how these genes interact with one another.Identifying these interactions within the genome can help us better understand the genetic architecture of complex traits, which in turn can lead to more personalized and precise treatments for many diseases.This is especially important because there is still much to learn about the heritability of many traits, and studying genetic interactions can help fill these gaps in our knowledge.

Table 1
Genetic results.
Illustration. 1. Gene expression networks associated with each other in the patient.N.Y.Madrid, L.J.M. Giraldo Journal of Genetic Engineering and Biotechnology 22 (2024) 100350